Conceptualizing Functional Traits and Ecological Characteristics of Methane-Oxidizing Bacteria

Conceptualizing functional traits and ecological characteristics of methane-oxidizing bacteria as life strategies.

Adrian Ho1,2, Frederiek - Maarten Kerckhof1, Claudia Luke2, Andreas Reim2,Sascha Krause3, Nico Boon1, and Paul L.E. Bodelier3*.

1Laboratory of Microbial Ecology and Technology (LabMET), Faculty of Bioscience Engineering, Coupure Links 653, B-9000 Ghent, Belgium.

2Max-Planck-Institute for Terrestrial Microbiology, Karl-von-Frisch-Str. 10, D-35043 Marburg, Germany.

3Microbial Ecology Department (NIOO-KNAW), Droevendaalsesteeg 10, 6708 PB, Wageningen, The Netherlands.

*Corresponding author

Running title: Functional traits of methane-oxidizing bacteria.

Keywords: Methane-oxidizing bacteria / functional traits/ microbial life strategies / microbial resource management.

Summary

Methane-oxidizing bacteria (MOB) possess the ability to use methane for energy generation and growth, thereby, providing a key ecosystem service that is highly relevant to the regulation of the global climate. MOB subgroups have different responses to key environmental controls, reflecting on their functional traits. Their unique features (C1-metabolism, unique lipids, and congruence between the 16S rRNA and pmoAgene phylogeny) have facilitated numerous environmental studies, which in combination with the availability of cultured representatives, yield the most comprehensive ecological picture of any known microbial functional guild. Here, we focus on the broad MOB subgroups (type I and type II MOB), and aim to conceptualize MOB functional traits and observational characteristics derived primarily from these environmental studies to be interpreted as microbial life strategies. We focus onthefunctional traits, and the conditions under which these traits will render different MOB subgroups a selective advantage. We hypothesize that type I and type II MOB generally have distinct life strategies, enabling them to predominate under different conditions and maintain functionality. The ecological characteristics implicated in their adopted life strategies are discussed, and incorporated into the Competitor-Stress tolerator-Ruderal(C-S-R) functional classification framework as put forward for plant communities. In this context, type I MOB can broadly be classified as competitor-ruderal (C-R) while type II MOB fit more within the stress tolerator categories. Finally, we provide an outlook on MOB applicationsby exemplifyingtwo approaches where their inferred life strategies could be exploited thereby, putting MOB into the context of microbial resource management.

Introduction: the role of MOB in the global methane cycle.

Methane is the third most important greenhouse gas, after water and carbon dioxide, contributing substantially to radiative forcing (Intergovernmental Panel on Climate Change, 2007). The atmospheric methane concentration has been increasing for most of the past century, followed by a stabilization in the past decade. Recently, atmospheric methane concentration continued to rise again (Rigby et al., 2008). The stabilizationhas been related to lower fossil fuel emissions (Bousquet et al., 2006), while the recent anomalies are linked to changes in microbial processes (Kai et al., 2011). Hence, the acquisition of knowledge regarding the underlying methane sources and sinks, including methane-oxidizing bacteria (MOB) demands immediate attention. Methane accumulation rates are determined by the balance of sources and sinks. The most important methane source, approximately 70% of the total budget of 500-600 Tg methane year-1,is the microbial production by methanogenic archaea in wetlands, areas associated with animal husbandry, and rice paddies (Intergovernmental Panel on Climate Change, 2007). The largest methane sink (> 80% of the total) is the photochemical reaction of methane with hydroxyl radicals in the troposphere, while diffusion of methane to the stratosphere and microbial methane oxidation account for the rest. Aerobic and nitrite-driven anaerobic methane oxidationare mediated by the MOB. A consortium seemingly comprised of methanogenic archaea and sulfate-reducing bacteria are thought to oxidize methane anaerobically in the marine ecosystem (Boetius et al., 2000; Orphan et al., 2002; Conrad, 2009; Orcutt et al., 2011). Recently, evidence was provided for the coupling of anaerobic methane oxidation to iron and manganese reduction in marine sediments (Beal et al., 2009), but the organism facilitating this process has not yet been isolated.In terrestrial environments, nitrite-driven anaerobic oxidation of methane may be an important methane sink, but is yet to be determined.To date, aerobic MOB are of high relevance to the global carbon cycle in terrestrial ecosystems, consuming atmospheric methane in non-flooded upland soils (Knief and Dunfield, 2005; Kolb, 2009), and attenuating methane emission from natural and anthropogenic wetlands (Brune et al., 2000). Therefore, aerobic MOB provide a key ecosystem service, mitigating up to 50% of biologically produced methane (Conrad, 2009).

The ability to use methane as a carbon and energy source is virtually restricted to MOB. Hence, if MOB activity is disturbed, functionality cannot be compensated by the action of other microbial groups, making biological methane oxidation a potentially vulnerable microbial community trait. Considering that MOB are comprised of subgroups with distinct ecology and functional traits, a shift in the MOB community composition or diversity may affect methane oxidation rates (Steenbergh et al., 2010; P.L.E. Bodelier, unpublished). Among atmospheric methane oxidizers (‘high-affinity’ MOB), MOB diversity is directly correlated to methane consumption, and lowers the variabilityof this process(Levine et al., 2011). MOB composition and activity, therefore, is fundamental to the observed fluctuations in methane consumption, and subsequent emission. However, the utilization of atmospheric methane is not universally distributed, but is associated with specificMOB groups (e.g. upland soil clusters; Knief et al., 2003) without any cultured representatives; hence, limiting our knowledge on their functional traits. We focus, instead, on the ‘low-affinity’ MOB, known to be active at >40 ppmv methane concentrations (Singh et al., 2010) which are detected in many high methane-emitting environments (e.g. rice paddies, landfills, lake sediments, and peatlands).The functional traits of MOB may mirror their life strategies. Here, we aim to determine these traits to conceptualize MOB life strategies for a better prediction of their response to environmental cues, and disturbances. Next, we determined whether this understanding could be applied to the context of microbial resource management.

Key players in methane oxidation in terrestrial ecosystems.

Traditionally, aerobic MOB group into type I and type II MOB belonging to gammaproteobacteria and alphaproteobacteria, respectively. Type I MOB, however, can befurther divided into type Ia MOB (e.g. Methylomonas, Methylobacter, Methylosarcina, and Methylomicrobium) and type Ib MOB (e.g. Methylococcusand Methylocaldum) based on the pmoA gene phylogeny (Bodrossy et al., 2003; Luke and Frenzel, 2011). Type I and type II MOB are distinguished in their phylogeny, physiology, biochemistry, and morphology (Trotsenko and Murrell, 2008; Semrau et al., 2010).Similarly, MOB can be identified based on their distinctive polar lipid-derived fatty acids (PLFA) patterns (Bodelier et al., 2009). Outside the canonical MOB, novel MOB belonging to Verrucomicrobiaand NC10 were recently discovered. Verrucomicrobial MOB are acidophilic, growing even at pH below 1 (Op den Camp et al., 2009), and may be prevalent in less hostile environments, but at present, their habitat range appears to be restricted to the environments from where they were isolated. The novel phylumNC10 is represented by a candidate bacterium Methylomirabilis oxyfera, capable of anaerobic methane oxidation coupled to denitrification (Ettwig et al., 2009; 2010; Strous, 2011). M. oxyfera apparently generates its own oxygen, subsequently used to oxidize methane. Of these three phyla, only proteobacterial MOB have been unequivocally provento be functionally important in natural and anthropogenic terrestrial environments: lake sediments (Dumont et al., 2011), rice paddies (Bodelier et al., 2000; Noll et al., 2008; Qiu et al., 2008), landfills (Chen et al., 2007), peatlands (Chen et al., 2008; Kip et al., 2010), high arctic wetlands (Graef et al., 2011), and floodplains (Bodelier et al., 2012).Henceforth, the general term MOB will be used to refer to aerobic proteobacterial MOB. However, with the discoveries of MOB belonging to novel phyla, efforts should be considered for their detection in futureenvironmental studies.

A moderately acidophilic MOB (optimum pH 5.0 - 5.5), Methylocella was demonstrated to grow on methane as well as other multicarbon compounds e.g. acetate, succinate, and pyruvate (Dedysh et al., 2005).Methylocella is not restrictedto acidic environments as previously thought;itsmmoX genewas detected in widespread environments with neutral or near neutral pH (e.g. rainforest soil, estuary sediment, Arctic soil, and rice paddy soil;Rahman et al., 2011; Reim et al., 2012).Although themmoX gene was retrieved from a rice paddy soil, corresponding transcripts could not be detected, suggesting that thesMMO plays only a marginal role – if any – oxidizing methane in this environment (Reimet al., 2012).Recently,Methylocystis spp. known to be an obligate MOB, have been shown to consume acetate and ethanol for growth (Belova et al., 2011; Im et al., 2011). These bacteria,and Methylocapsa, also a proven facultative MOB (Dunfield et al., 2010), fall into alphaproteobacteria that use the serine cycle for carbon assimilation, while gammaproteobacterial MOB assimilate carbon via the ribulose monophosphate pathway (Semrau et al., 2010). Methylocella and Methylocapsabelong toBeijerinckiaceae, but possess cytological and biochemical similarities with Methylocystis. Interestingly, facultative MOB appear to be confined to the alphaproteobacteria,suggesting a more versatile substrate utilizationthan inthe gammaproteobacterial MOB, and render them a survival strategy when methane availability is limited or fluctuates.

Discoveries of novel microorganisms oxidizing methane have pushed the boundary of MOB phylogeny. Therefore, the provisional grouping of MOB into type Ia MOB, type Ib MOB, and type II MOB, although still acceptable for proteobacterial MOB at present, may change in future. The key enzyme for methane oxidation is the methane monooxygenase (MMO), existing either as a particulate membrane bound (pMMO) or soluble (sMMO) form. Virtually all MOB possess the pMMO, with the exception of Methylocella and Methyloferula (Dedysh et al., 2000; Vorobev et al., 2011), while the sMMO is confined to some MOB. Copper regulates the expression of MMO in MOB that possess genes for both forms of the enzyme, stimulating the pMMO expression at high copper to biomass ratio, while repressing the sMMO (Stanley et al., 1983; Murrell et al., 2000; Knapp et al., 2007). The pmoA gene, present in duplicate copies in some MOB (Semrau et al., 1995), encodes for the β-subunit of the pMMO enzyme, is highly conserved, and has been generally found to correspond to the 16S rRNA gene phylogeny (Kolb et al., 2003), making pmoA an alternative to the 16S rRNA gene, and a suitable marker for culture-independent studies (McDonald et al., 2008).

Environmental control of MOB.

Methane

Abiotic environmental factors affecting methane oxidation and the MOB have been reviewed (Conrad, 2007; Semrau et al., 2010). Among these, methane concentration and nitrogen availability arethe most well studied factors, and are strong driving forces shaping MOB community composition and activity, asserting different responses in type I and type II MOB. A comprehensive list detailing MOB ecological characteristics possibly differentiating the functional traits belonging to type I and type II MOB are summarized (Table 1).Recently, a novel isoenzyme, pMMO2, was found in a MOB, and seems to be restricted within the type II Methylocystis-Methylosinus group (Yimga et al., 2003; Baani and Liesack, 2008).pMMO2 allows MOB to grow at low methane concentrations (<100 ppmv),but growth was not detected at atmospheric methane levels, whereas the conventional pMMO is typically expressed under higher methane concentrations (>600 ppmv). Hence, some type II MOB may possess an advantage under methane depleted conditions,having the ability to withstand methane fluctuations. On the other hand, the ‘low-affinity’ MOB are found in many methane-emitting environments, and are represented by both type I and type II MOB.

Nitrogen

It was generally accepted that nitrogen fertilization had an inhibitory effect on methane oxidation, probably through competitive inhibition of the MMO by ammonia (Gulledge and Schimel, 1998; Bodelier and Laanbroek, 2004). However, Bodelier and colleagues (2000) found a stimulation of MOB activity and growth upon ammonium fertilization in a rice microcosm. Upon relief of nitrogen limiting conditions, MOB responded rapidly (within minutes) to nitrogen addition (Bodelier et al., 2000), suggesting a more direct mechanism affecting the MOB metabolism (Bodelier and Laanbroek, 2004). However, the effects of ammonium were not clear in a soil and ricemicrocosm study, respectively (Shrestha et al., 2010; Krause et al., 2012). Although repeatedly examined, the response of MOB activity to ammonium amendment is inconsistent, showing inhibition, stimulation, or no effect, suggesting that the variability observed was attributable to theinherent characteristics of the MOB composition, or the ammonium load tested. On the other hand, nitrite had been shown to differentially affect MOB, making it a potential inhibitory compound, particularly for type II MOB (Nyerges et al., 2010). Generally, nitrite exerts a toxic effect that leads to inhibition of methane uptake (Schnell and King, 1994), and is known to inhibit formate dehydrogenase (Jollie and Lipscomb, 1991). However, these effects are studied with pure cultures whereas under field conditions, the ability to denitrify (Campbell et al., 2011) may aid MOB to detoxify nitrite. The nifH gene encoding for the enzyme nitrogenase reductase was detected inboth type I and type II MOB, but nitrogen fixation seems to be a characteristic ofmainly type II MOB (Murrell and Dalton, 1983; Auman et al., 2001). At the community level, ammonium amendment was shown to selectively stimulate type I MOB in a rice paddy and forest soil, respectively (Bodelier et al., 2000; Mohanty et al., 2006; Noll et al., 2008). Although activity may vary, it is becoming clear that MOB subgroups respond differently to nitrogen availability, indicating their level of tolerance to or dependency onnitrogen amendments.

Life strategies: type I and type II MOB.

Accumulating evidence concerning the ecological characteristics of type I and type II MOB, and community level molecular analyses of MOB populations under different conditionssuggest that the different MOB subgroupspossess distinct traits, reflecting on their life strategies (Table 1). The detection of marker genes for MMO (e.g. pmoA,mmoX)is central for many molecular analysesand indicates the potential active community, taking into account the current and previous members contributing to the MOB seed bank, while retrieval of the corresponding gene transcript (mRNA) is typically considered to be a proxy for activity, and suggest the active population (Jones and Lennon, 2010). Experiments using stable isotope labeling, however, provide a direct link between function and microbial identity (Dumont and Murrell, 2005). Based on stable isotope (13C-methane) labeling experiments, an apparent emerging pattern shows that type I MOB, although numerically less dominant than type II MOB, are predominantly active in many important habitats with high methane emissions (Chen et al., 2007; Noll et al., 2008; Qiu et al., 2008; Kip et al., 2010; Dumont et al., 2011; Graef et al., 2011). Moreover, type I MOB (Methylobacter) have been shown to be indicative of environments with a high methane source strength (Krause et al., 2012), and was predominant in an Arctic tundra soil where virtually only type IMOB was detected (Liebner et al., 2009). Further evidence was demonstrated by Ho and colleagues (2011a), showing that the higher potential for methane oxidation corresponded well,particularly to the growth and activity of type Ib MOB in a rice paddy soil. Using soil from a river floodplain, incubations under methane showed a biphasic depletion curve of ‘initial’ and ‘induced’ uptake rates (Steenbergh et al., 2010). The ‘initial’ phase isgenerally considered to represent in-situ oxidation rates, whereas the ‘induced’ phase was shown to be contributed by an increase in MOB cell numbers and cell specific activity. Regardless, in both phases, the pmoA gene expression level and growth rates were significantly higher for type I MOB. Despite of the diverse environments, these studies provide strong evidence that generally, type I MOB are very responsive to high substrate availability, but when conditions are limiting or adverse, numbers are reduced quickly.

On the contrary, it is thought that the type II MOB populationis relatively stable, and assumed to be present in a dormant state forming part of the microbial seed bank in the soil (Eller et al., 2005; Krause et al., 2012). Indeed, type II MOB generally forms more desiccation- and heat-resistant resting cells than type I MOB (Whittenbury et al., 1970). Here, we define dormancy as a state of reversible reduced metabolic activity and can be discriminated by not being able to detect the population at the gene transcription (mRNA) level. Accordingly, while the pmoA gene belonging to type II MOB was detected, the corresponding transcript was not retrieved or retrieved in relatively low levels, suggesting their presence, but inactive role in the soil (Bodrossy et al., 2006; Krause et al., 2010). Although largely dormant, type II MOB became more important during recovery from disturbances or under fluctuating conditions. Results show that upon a disturbance-induced die-off, type II MOB populationincreased, and dominated the total MOB population after 40 days, while type I MOB showed a rapid response soon after the disturbance (Ho et al., 2011b). The initial relatively higher nutrient availability may have sustained type IMOB dominance (Mohanty et al., 2006; Krause et al.,2010), but type II MOB, being less demanding, became more competitive later when nutrients were limiting (Graham et al., 1993). In another form of disturbance, the type II population numerically increased after a brief exposure to heat stress at 45°C, and subsequently led to a higher methane uptake rate than in the control (continuous incubation at 25°C) (Ho and Frenzel, 2012). Hence, it was suggested that a brief exposure to elevated temperatures may have triggered the translation of type II MOB from dormant to metabolically active states (Whittenbury et al., 1970; Ho and Frenzel, 2012).Nevertheless, methane uptake was significantly lower in prolonged incubations at temperatures exceeding 40°C, likely due to the decreased activity of mesophilic MOB (Mohanty et al., 2007). Despite of the different disturbances simulated, type II MOB were persistent and recovered well, andappear to have a different adaptation strategyfrom type I MOB.

The traits of type I and type II MOB observed so far have often been interpreted as a reflection of the r- and k-selection theory (Steenbergh et al., 2009, Siljanen et al., 2011; Bodelier et al., 2012) , designating organisms to be evolutionary r-selected that invest in high reproductive success, and short life spans being most effective in unstable environments. K-selectedorganisms invest in maintaining numbers at carrying capacity of the habitat, having low off-spring and growth rates typically displayed in stable habitats (MacArthur and Wilson, 1967). However, considering the knowledge gathered so far (see table 1), this 2-dimensional framework is designed for animal life-strategies, and do not represent MOB life strategies in an accurate way. The long-term survival of microbes under adverse conditions, their limited mobility in combination with their potential emergence from microbial seed banks makes their life strategies more similar to plants than animals.